Exosomal miR‐21‐5p derived from bone marrow mesenchymal stem cells promote osteosarcoma cell proliferation and invasion by targeting PIK3R1

Abstract Mesenchymal stem cells (MSCs) are a class of pluripotent cells that can release a large number of exosomes which act as paracrine mediators in tumour‐associated microenvironment. However, the role of MSC‐derived exosomes in pathogenesis and progression of cancer cells especially osteosarcoma has not been thoroughly clarified until now. In this study, we established a co‐culture model for human bone marrow‐derived MSCs with osteosarcoma cells, then extraction of exosomes from induced MSCs and study the role of MSC‐derived exosomes in the progression of osteosarcoma cell. The aim of this study was to address potential cell biological effects between MSCs and osteosarcoma cells. The results showed that MSC‐derived exosomes can significantly promote osteosarcoma cells’ proliferation and invasion. We also found that miR‐21‐5p was significantly over‐expressed in MSCs and MSC‐derived exosomes by quantitative real‐time polymerase chain reaction (qRT‐PCR), compared with human foetal osteoblastic cells hFOB1.19. MSC‐derived exosomes transfected with miR‐21‐5p could significantly enhance the proliferation and invasion of osteosarcoma cells in vitro and in vivo. Bioinformatics analysis and dual‐luciferase reporter gene assays validated the targeted relationship between exosomal miR‐21‐5p and PIK3R1; we further demonstrated that miR‐21‐5p‐abundant exosomes derived human bone marrow MSCs could activate PI3K/Akt/mTOR pathway by suppressing PIK3R1 expression in osteosarcoma cells. In summary, our study provides new insights into the interaction between human bone marrow MSCs and osteosarcoma cells in tumour‐associated microenvironment.


| INTRODUC TI ON
Osteosarcoma (OS) is one of the most common primary malignant and aggressive type of bone cancer in adolescents. Recurrence and metastasis are the main reasons for the unsatisfactory treatment of OS. 1 However, the mechanism of proliferation and invasion of OS is still not clear. As we all know, the biological behaviour of cancer is not only determined by cancer cells alone, and stromal cell in tumour-associated microenvironment acts on cancer cells to promote cancer proliferation and invasion, which has become more and more of a concern. As a non-tumour cell, mesenchymal stem cells (MSCs) may participate in processes of OS proliferation and invasion in OS-associated microenvironment. 2 MSCs are a kind of nonhematopoietic progenitor cells with multipotent and self-renewable abilities which reside in bone marrow, umbilical cord, placenta tissue, adipose tissue, etc. [3][4][5][6] Furthermore, MSCs have biological characteristics of migrating to the cancer site where they participate in the formation of tumour-associated microenvironment and have always been studied in the field of cancer pathogenesis and cancer therapy. 7,8 MSCs not only provide the microenvironment for cancer cells, but also enhance cancer progression and invasion. 9 It has been found that the aneuploidy and genomic loss can transform MSCs into OS cells, indicating that OS cells may originate from bone marrow MSCs. 10 Studying the role of MSCs in OS proliferation and invasion is of great significance to elucidate the mechanism of OS progression.
Classically, MSCs play its role mainly through cell contactdependent mechanisms and soluble factors. 11 However, further studies have found that MSCs could secrete a large number of exosomes through paracrine mechanism, which can change the microenvironment around target cells, thus regulating the biological functions of cell proliferation and differentiation. [12][13][14] Exosomes are small, lipid bilayer membrane vesicles with a diameter of about 30-100 nm and contain a lot of specific biologically active molecules such as nucleic acids, lipid, and proteins. 15 Moreover, exosomes carry a variety of types of RNA, DNA, protein and other signal molecules of donor cells. 16 Its main biological function is to transmit the signal molecules to recipient cells, thus forming the information exchange and transmission between cells. 17 In addition, exosomes are involved in the pathological processes of various diseases, including cancer. 18 For example, studies have shown exosomes from different cell types involved in cancer cells proliferation, angiogenesis, differentiation and metastasis. [19][20][21][22] In previous studies, we found that MSC-derived exosomes enhance the proliferation of OS, 23 but we have not yet elucidated the specific molecular biological mechanism. Recently, it has been found that the expression of miR-21-5p increased during the osteogenic differentiation of MSCs. 24 MiR-21-5p is considered to be an oncogene that promotes tumour proliferation and invasion. 25 Here, we found that MSC-derived exosomes may be rich in miR-21-5p, which further affects the biological characteristics of OS, and the exosomes protect miR-21-5p from RNase degradation in OS-associated microenvironment. Bioinformatics prediction found that PIK3R1 is the target gene of miR-21-5p and is also responsible for encoding an important regulatory subunit (p85α) of phosphatidylinositol 3-kinases(PI3K), PI3K forms a heterodimeric protein complex composed of p85 regulatory subunit and catalytic p110 subunit encoded by the PIK3CA. 26,27 Furthermore, PIK3CA oncogene carrying hotspot mutation has carcinogenic activity. In contrast, PIK3R1 seems to play a tumour-suppressor role because PI3K subunit p85α adjusts and stabilizes p110. [27][28][29] P85 type also has other two subunits: PIK3R2 and PIK3R3. Classically, p85α and p85β are considered to be similar proteins associated with activated receptor tyrosine kinases (RTK) that induce PI3K activation; however, PIK3R1/p85α is the most abundant subtype in tumour-free tissues, but its expression is reduced in cancer, which has a tumour-suppressor function. 30 In contrast, PIK3R2/p85β expression is upregulated in cancer which is regarded as a tumour driver. 31-33 PI3K activation is induced by p85 binding to activated receptor tyrosine kinase (RTK). 34 PI3K/Akt/ mTOR is the signalling pathways closely related to apoptosis and cell proliferation, which is usually highly activated in OS. 35

| Identification of MSCS-and MSCderived exosomes
Cellular surface antigens of MSCs were examined with flow cytometry, and it has been described in detail in previous experiment. 36 The osteogenic and adipogenic differentiation of MSCs was further performed by using the differentiation media and detected by Oil red O staining and Alizarin red staining (Cyagen Bioscience, Inc.). Cellular surface antigens were examined by using a flow cytometry of MSCs   for CD19, CD29, CD90, CD44, CD73, CD105 and CD133 markers. MSC-derived exosomes were isolated according to the exosome extraction protocol. 37 In short, cell culture supernatants at 300 × g centrifugation for 10 min to remove cells, to 2000 × g centrifuged for 10 min to remove dead cells, and at 10,000 × g for 30 min to remove cell debris and retain the supernatant for further ultracentrifugation.
At twice 100,000 × g ultracentrifugation for 70 min, the pellets (exosomes) were retained, and the supernatant was discarded. Purified exosomes were negatively stained with uranyl acetate by means of floating method and observed by transmission electron microscope.
The particle size distribution of exosomes was characterized and quantified by NanoSight LM10 system (NanoSight).

| Co-culture experiments
MSCs were seeded in a 6-well plate of a density of 1 × 10 4 cells/cm 2 , and OS cells seeded in 6-well chambers with 0.4 μm polycarbonate membrane pores. The membrane was permeable, and cells could not pass through the membrane, but the cytokines secreted by cells could pass through. Cells were co-cultured for 2 weeks, and then, induced MSCs were collected for further experiments.

| Choice of differentially expressed mirnas list using heat map analysis
We obtained the microarray data from Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/), and the GEO accession Nos. are GSE58027 and GSE89930. The heat map of miRNAs showed significant differences using the hierarchical clustering method. 38

| Data mining in oncomine database
Oncomine database (https://www.oncom ine.org/resou rce/login. html) is a publicly accessible online cancer microarray database, which helps to promote the research of genome-wide expression analysis. 39 We used Oncomine database to determine the transcription level of PIK3R1 gene in sarcoma by searching the expression level of PIK3R1 mRNA (log2 transformation) in sarcoma and normal tissues (tumour-free tissues).

| PKH26-labelled exosome and confocal microscopy
PKH26 was used to label exosomes, and 4′,6-diamidino-2-phenylindole (DAPI) was used to label OS cell nuclei. The process of OS cells uptake of exosomes was observed under a Nikon Eclipse 80i confocal fluorescence microscope.

| Treatment of MSCs with GW4896
For the inhibition of exosome generation, MSCs were pre-treated with exosome-free media containing 10 μM GW4869 (Umibio) for 24-48 h. When wall-adhered MSCs reached 90% confluence, the culture supernatants were collected for further cell proliferation assays.

| Wound-healing assay
OS cells were divided into a 6-well plate at a rate of 1 × 10 5 cells per well. When cells grew to 95% confluence, monolayers were wounded by a sterile 10 μl plastic micropipette tip, washed and added different culture medium according to the needs of the experiment. The width of the scratch gap under the microscope was observed and photographed.

| Western blotting
Protein extraction and Western blotting were performed as described previously. 43 The following antibodies were used according to the manufacturer's instructions: Calreticulin, CD63 (Abcam, Inc.).
Proteins were detected using specific antibodies (Abcam, Inc.). The expressions of Bcl-2, Bax, PIK3R1, PIK3R2 and PI3K/AKT/mTOR pathway-related proteins in human OS cell lines were also assessed by WB assays. The primary antibodies including Bcl-2, Bax, PIK3R1, PIK3R2, phosphorylated (p)-PI3K, PI3K, p-Akt, Akt, p-mTOR, Mtor and β-actin were used as a loading control. through subcutaneous injection. After 1 week of tumour growth, mice were randomly divided into the following groups (n = 6): OS cells mixed with miR-21-5p-exosomes (MSCs were treated with miR-21-5p mimic to purify miR-21-5p-exosomes) and OS cells mixed with miR-NC-exosomes. In control mice, we injected intra-tumour, the same volume of PBS (vehicle) alone. Each mice (n = 6/group) was injected with MSC-derived exosomes suspension (400 μg/ml) by intra-tumour injection every week. After injection, calliper was used to measure the tumour size, and the following formula was used to calculate the tumour volume: Tumour volume (mm 3 ) = 0.52 × width (mm) 2 × length (mm). At the end of the 5th week, nude mice were sacrificed using 20% overdose pentobarbital.

| Statistical analyses
The data were presented as mean ± standard deviation and statisti-

| Characterization of MSCs-and MSC-derived exosomes
MSCs from bone marrow of patients diagnosed with OS began to adhere to the wall after inoculation 24 h, and after 2-3 passages, MSCs displayed fibroblast-like cell adherent growth and grew in a long spindle shape ( Figure 1A). Flow cytometry showed that MSCs expressed CD29, CD90, CD44, CD73 and CD105 markers, but negative results for CD19 and CD133 ( Figure 1B). After 2 weeks of osteogenic and lipogenic induction, MSCs differentiated into osteocytes and adipocytes, stained with alizarin red S and oil red ( Figure 1C).
Transmission electron microscopy showed that a large number of MSC-derived exosomes were typical small spherical nanoparticles with a diameter of 40-80nm ( Figure 1D). NanoSight analysis showed the diameter distribution of exosomes ranged from 30 to 165 nm, and exosomes with diameter of 75 nm had a peak ( Figure 1E).
MSC-derived exosomes express primarily surface marker CD63, and Calreticulin, an intracellular protein, was negatively expressed ( Figure 1F). Calreticulin is a highly conserved chaperone protein of the endoplasmic reticulum that has specificity towards glycoprotein substrates, which exists in all cells except red blood cells in highgrade biology. 44

| Characterization of exosome internalization by OS cells
To investigate whether MSC-derived exosomes were internalized by OS cells, we used the fluorescent dye, PKH26, to labelled MSCderived exosomes, and OS cell nuclei were stained by DAPI. Under the confocal laser microscope, most of OS cells could be observed in a red fluorescence signal, the fluorescent exosomes are mainly located in the cytoplasm ofU2OS and MG63. In order to eliminate PKH26 dye contamination in U2OS and MG63, exosome-free supernatant was also stained by PKH26 and no red fluorescence was detected ( Figure 2).

21-5p and targeting gene PIK3R1
After co-culture of MSC-derived exosomes with OS cell, the proliferation and invasion of OS cells increased. We predicted that MSC-

| Exosomal MIR-21-5p derived from MSCs can accelerate OS cell proliferation and invasion
To was much lower compared with that in the normal group (p < 0.05).

| Exosomal MIR-21-5p derived from MSCs promote tumour growth in vivo
To study the effect of exosomal miR-21-5p derived from MSCs on  Figure 7A). In the 4th week, intra-tumour injection of miR-21-5p-exosomes significantly increased the tumour volume and weight compared with miR-NC-exosomes and vehicle-treated control (p < 0.05, Figure 7A). At the sacrifice stage, the tumour volume and weight were significantly greater in mice treated with miR-21-5p-exosomes than in controls injected with miR-NC-exosomes or with the vehicle alone (p < 0.05, Figure 7A,B).
In addition, Western blot analysis found that miR-21-5p inhibition of MSC-derived exosomes enhanced PIK3R1 protein levels, and miR-21-5p overexpression of MSC-derived exosomes induced the opposite effects in U2OS and MG63 cells. However, there were no significant differences in PIK3R2 expression between groups ( Figure 8B). The results of RT-PCR showed that the expression level of PIK3R1 has no statistical difference between miR-21-5p inhibitor group and miR-21-5p mimics group ( Figure 8C). This suggested that miR-21-5p inhibits the expression of PIK3R1 at post-transcriptional level.
To gain further mechanistic insights into the roles of miR-21-5p/ PIK3R1 axis, we hypothesized that exosomal miR-21-5p derived from MSCs activates the PI3K/Akt/mTOR signalling pathway in OS, leading to OS proliferation and invasion, because the PI3K regulatory subunit p85α/PIK3R1 can exert a tumour-suppressor effect by were relatively stable in OS cells ( Figure 8D).
In summary, the results indicated that bone marrow MSCs could produce amounts of exosomes, which may deliver miR-21-5p into OS cells and thus promote OS proliferation and invasion. The increased miR-21-5p in OS cells specifically targeted the 3′UTR region of PIK3R1 mRNA, leading to the decrease in p85α protein level and the activation of corresponding PI3K/Akt/mTOR signalling pathway.
A detailed summary of the results is showed in Figure 9.

| DISCUSS ION
Recently, more and more studies have shown that exosomes play a vital role in information transfer between cells 16  understand the potential molecular mechanism on OS.
The extraction of exosomes was the main difficulty of this study.
The main methods of exosome separation and extraction are gradient ultracentrifugation, kit extraction, ultrafiltration, density gradient centrifugation, polymeric precipitation and immunomagnetic beads. 54 In this experiment, we purified MSC-derived exosomes by gradient ultracentrifugation, which has the advantage of high purity. However, this method is complicated, costly and time-consuming and it requires the laboratory to have an ultra-speed centrifuge and its corresponding centrifugal rotor. We found that after OS cells were co-cultured The Western blot assay showed the protein expression level of PIK3R1 was reduced by miR-21-5p overexpression of MSC-derived exosomes inU2OS and MG63 cells. β-actin was used as an internal control. (C) RT-PCR showed the expression level of PIK3R1 has no statistical difference between miR-21-5p inhibitor groups and miR-21-5p mimics groups. (D) Western blot assay was implemented to measure the expression levels of p-or not PI3K, Akt and mTOR in U2OS and MG63 after treatment vs normal group. All experiments were implemented in triplicate, *p < 0.05, ***p < 0.001. mt, mutant; NC, negative control; p-, phosphorylated; wt, wild type membrane changes and activate downstream caspase-3 cascade reaction when cells are exposed to apoptosis signal and then induce apoptosis. 56 To further study the mechanism of MSC-derived exosomes on OS cell proliferation and invasion, we found that in OS-associated microenvironment, MSC-derived exosomes express high level of miR-21-5p, which can be regarded as oncogene in cancer. 25,57 Moreover, study have found that miR-21-5p is over-expressed in various types of tumours, and it plays an important role in tumorigenesis and tumour progression; high miR-21-5p expression is related to poor prognosis and short survival. 25 Does MSCs in OS-associated microenvironment involved in OS proliferation and invasion by mediating exosomal miR-21-5p? At present, it has not been studied.
We found transfection of miR-21-5p mimics or miR-21-5p inhibitor into MSCs could significantly up-or downregulate the expression of miR-21-5p in OS cells and can significantly promote or attenuate the proliferation and invasion of OS. High expression of miR-21-5p in primary OS can be used as an indicator of diagnosis and prognosis of OS. 58 Importantly, we firstly found MSC-derived exosomal miR-

21-5p promotes OS proliferation and invasion by targeting PIK3R1
and activity of cancer-related PI3K/Akt/mTOR signalling pathway.
This study is the first to investigate the relationship between MSC-exosomal miR-21-5p and targeting gene PIK3R1 in OS. Our study has revealed that PIK3R1 is the only down-expressed member of PI3K in OS. PI3K is a key molecule in the PI3K/Akt/mTOR signalling pathway, which is involved in the regulation of a variety of transcription factors in cancer. 59 PI3K is a heterodimer composed of a regulatory subunit (p85) and a catalytic subunit (P110). The regulatory subunit (p85) contains SH2 and SH3 domains and interacts with target proteins containing corresponding binding sites.
According to the different regulatory subunits, class I PI3Ks are divided into three categories, with different structures and functions. 60 PIK3R1 gene encode one of p85-type subunits: p85α. 30 PIK3R1/p85α is abundant isoform in tumour-free tissues, but its expression is reduced in tumour cells. 31

ACK N OWLED G EM ENTS
The authors would like to thank Yali Zhou and Yali Liu for flow cytometry. Flow cytometry assays were performed on a device of Beckman coulter.
F I G U R E 9 Schematic of the action of exosomal miR-21-5p derived from human bone marrow MSCs regulating OS cells progression. Human bone marrow MSCs transmit miR-21-5p to OS cells through exosomes, exosomal miR-21-5p target PIK3R1 gene of OS cells, then activating PI3K/Akt/mTOR signal pathway and leading to OS proliferation, invasion and anti-apoptosis